Wrighton DC et al. (2015), Mechanism of inhibition of mouse Slo3 (KCa 5.1)...

XB-ART-50796

Br J Pharmacol 2015 Sep 01;17217:4355-63. doi: 10.1111/bph.13214.

Show Gene links Show Anatomy links

Mechanism of inhibition of mouse Slo3 (KCa 5.1) potassium channels by quinine, quinidine and barium.

Wrighton DC , Muench SP , Lippiat JD .

???displayArticle.abstract???
BACKGROUND AND PURPOSE: The Slo3 (KCa 5.1) channel is a major component of mammalian KSper (sperm potassium conductance) channels and inhibition of these channels by quinine and barium alters sperm motility. The aim of this investigation was to determine the mechanism by which these drugs inhibit Slo3 channels. EXPERIMENTAL APPROACH: Mouse (m) Slo3 (KCa 5.1) channels or mutant forms were expressed in Xenopus oocytes and currents recorded with 2-electrode voltage-clamp. Gain-of-function mSlo3 mutations were used to explore the state-dependence of the inhibition. The interaction between quinidine and mSlo3 channels was modelled by in silico docking. KEY RESULTS: Several drugs known to block KSper also affected mSlo3 channels with similar levels of inhibition. The inhibition induced by extracellular barium was prevented by increasing the extracellular potassium concentration. R196Q and F304Y mutations in the mSlo3 voltage sensor and pore, respectively, both increased channel activity. The F304Y mutation did not alter the effects of barium, but increased the potency of inhibition by both quinine and quinidine approximately 10-fold; this effect was not observed with the R196Q mutation. CONCLUSIONS AND IMPLICATIONS: Block of mSlo3 channels by quinine, quinidine and barium is not state-dependent. Barium inhibits mSlo3 outside the cell by interacting with the selectivity filter, whereas quinine and quinidine act from the inside, by binding in a hydrophobic pocket formed by the S6 segment of each subunit. Furthermore, we propose that the Slo3 channel activation gate lies deep within the pore between F304 in the S6 segment and the selectivity filter.

???displayArticle.pubmedLink??? 26045093
???displayArticle.pmcLink??? PMC4556473
???displayArticle.link??? Br J Pharmacol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis

???attribute.lit??? ???displayArticles.show???

	Figure 1. Expression of WT, R196Q and F304Y mSlo3 K+ channels in Xenopus oocytes. (A) Representative current families recorded by two electrode voltage clamp from oocytes injected with different mSlo3 RNA or with no RNA (control) as indicated. Oocytes were held at −80 mV and 100 ms pulses to potentials between −100 and +140 mV were applied. The dashed line represents the zero-current levels and scale bars represent equivalent current amplitudes and timescales. (B) Mean (± SEM) current–voltage relationships of oocytes expressing WT mSlo3 (WT, n = 29), R196Q mSlo3 (n = 8) F304Y mSlo3 (n = 26), and non-injected oocytes (control, n = 12). For symbols used see part (A). (C) Mean (± SEM) resting membrane potential of oocytes in standard Ringer's solution. *P < 0.0001 compared with control oocytes (Kruskal–Wallis test).
	Figure 2. Block of mSlo3 currents by inhibitors of KSper. Oocytes were held at −80 mV and depolarizing pulses to +100 mV were applied. (A) Representative traces with each drug or condition as indicated: black trace, control currents recorded prior to drug application; light grey trace, current in the presence of the inhibitor; dark grey trace, current after washing out the inhibitor for at least 10 min. The dashed line represents the zero-current level and scale bars represent equivalent current amplitudes and timescales. The effects of Ba2+ were measured in both the standard solution containing 2.5 mM K+ (2.5K) and with a high 100 mM K+ solution (100K). (B) Mean percentage inhibition for each drug or condition as indicated (n values indicated in parentheses in the bars).
	Figure 3. Concentration-dependent inhibition of WT, F304Y and R196Q mSlo3 currents by quinine and quinidine. Representative traces recorded before (0) and in the presence of quinine (A) and quinidine (B) (concentrations in μM as indicated). The dashed line represents the zero-current levels and scale bars represent equivalent current amplitudes and timescales. Mean (± SEM) concentration–inhibition plots for quinine (C) and quinidine (D) inhibition of WT mSlo3, R196Q mSlo3 and F304Y mSlo3 at +100 mV, fitted by the Hill equation provided in the Methods. Apparent voltage-dependence of the IC50 for quinine (E) and quinidine (F) of WT and F304Y mSlo3 currents. The data are described and analysed further in the main text. For key to symbols used see Figure 1.
	Figure 4. Concentration-dependent inhibition of WT and F304Y mSlo3 currents by Ba2+. (A) Representative traces recorded before (0) and in the presence of Ba2+ (concentrations in mM as indicated). The dashed line represents the zero-current levels and scale bars represent equivalent current amplitudes and timescales. (B) Mean (± SEM) concentration inhibition plots for WT and F304Y mSlo3 at +100 mV, fitted by the Hill equation provided in the Methods. (C) Apparent voltage-dependence of the IC50 for block by Ba2+ of WT and F304Y mSlo3-mediated currents. The data are described and analysed further in the main text. For key to symbols used see Figure 1.
	Figure 5. Molecular modelling of mSlo3 and inhibition by quinidine. (A) Homology model of the transmembrane regions S1 to S6 of mSlo3; the structure is shown as a side-on view from the membrane, with the extracellular space above the protein. Each subunit of the tetrameric structure is coloured differently for clarity and only side chains F304, I308 and V312 are shown in stick format. (B) A zoomed-in view of the proposed quinidine-binding site, with subunits labelled blue, red and green, (the fourth subunit has been removed for clarity). The residues which make up the hydrophobic binding pocket (F304, I308 and V312) are shown in stick format and the surface shows the hydrophobic isoelectric character. (C) The same view as in (B) but with tyrosine replacing F304, showing the proximity of the terminal oxygen to the methoxyquinoline group of quinidine where hydrogen bonding could occur. In both (B) and (C), the quinidine molecule is shown in stick format and coloured, yellow, blue and red for carbon, nitrogen and oxygen respectively. (D) Proposed mechanisms for the inhibition induced by Ba2+ ions and quinine or quinidine (Qn/Qd): Ba2+ enters the pore from the extracellular side and blocks at the selectivity filter; quinine and quinidine cross the membrane and block from the intracellular side at a site involving F304 and I308 (left). In the F304Y mutation (right), the Ba2+ block is unaffected, but a deeper binding site is available for quinine and quinidine entering the pore from the intracellular side. Possible mechanisms are discussed in the main text.

References [+] :

Alexander, The Concise Guide to PHARMACOLOGY 2013/14: ion channels. 2013, Pubmed